The Effect Of IDH1-R132H Mutation In Glioma On The Antitumor Response Of NK Cells

ObjectiveGlioma originates from the mutation of glial cells in the brain and spinal cord,and it’s the most common tumor in central nervous system.Glioma has the worst prognosis in human tumors with less than 2 years median survival time as well as increasing incidence in recent years.Surgery is the main treatment of glioma,but it is hard to reset the tumor tissue completely.Although chemotherapy and radiotherapy have a certain promising effect,but they also had some side effects,limiting the curative effect,thus,glioma still has a high rate of metastasis and recurrence,leading an unfavorable outcome.Therefore,it is crucial to investigate the mechanisms of occurrence and development of glioma,and find new targets and strategies for the treatment of glioma.Since 2008,Parsons et al.first detected IDH1(isocitric dehydrogenase gene 1)mutations in glioma by high-throughout sequencing.There has been continuous research found that the mutation is highly corelated with diagnosis,treatment as well as the evaluation of the glioma patient’s prognosis.Thus,investigation of IDH1 gene mutations has become one of hotspots in the study of glioma.At present,many studies have confirmed that the mutations of IDH1 gene,especially the R132 H mutation,can significantly improve the prognosis of the glioma patients.However,the molecular mechanisms were not yet clear.Previous studies have been reported that IDH1 mutations affect the patient’s prognosis mainly through metabolic processes,while the effects of IDH1 mutations on the immune microenvironment of glioma are still poorly understood.The tumor microenvironment mainly refers to the complex environment,which maintains the growth of tumor,and includes tumor cells,cytokines,chemokines,stromal cells,etc during the process of tumor occurrence and progression.Tumor microenvironment,especially the tumor immune microenvironment,immune cells and molecules that inhibit or promote tumor growth in tumor environment,also plays an important role in tumor occurrence,development,metastasis and prognosis.This study aimed to investigate how IDH1-R132 H mutation affects glioma immune microenvironment and improves the prognosis of glioma patients,and ultimately provide potential therapeutic target for tumor immunotherapy of patients with IDH1-R132 H mutation.MethodsTCGA and CGGA databases were used to analysis the difference of prognosis between IDH1 wild type(IDH1-WT)and IDH1-R132 H mutant glioma(IDH1-R132H)patients and screen immune cells and cytokines expressed differently in two groups,clinical tissue samples were collected for validation;Kaplan-Meier survival analysis was used to analysis the correlation between CD56 expression and survival of patients;IDH1-WT and IDH1-R132 H heterozygous mutant cell lines were constructed in vitro,and verified by Western-Blot and mass spectrometry;NK cells derived from health doors were expanded in vitro and co-cultured with IDH1-WT and IDH1-R132 H cell lines;transwell assay was used to validate the difference of recruiting NK cells between the two cell lines;clinical tissue samples and public databases were used to screen chemokines related to recruiting NK cells,q-PCR and Western-Blot were used to validate the differences at cell line level;flow cytometry was used to analysis the difference of CX3CR1 expression,the CX3CL1 receptor,between IDH1-WT and IDH1-R132 H in clinical sample tissues;the mechanisms of IDH1-R132 H induced upregulation of CX3CL1 was tested by Western-Blot;the relationships between CX3CL1 and CD56 expression and prognosis were evaluated by analyzing clinical samples and public databases both in whole glioma and IDH1-R132 H glioma patients;finally,CRISPR-Cas9 mediated genome editing technology was used to knockout background expressed IDH1 gene in U87 cell line,and constructed the IDH1-R132 H homozygous mutant cell line.Results1.TCGA and CGGA databases analyzing revealed that IDH1-R132 H mutation improved prognosis of glioma patients.2.We constructed plasmids with overexpressed IDH1-WT and IDH1-R132 H,and IDH1 wild type and IDH1-R132 H heterozygous mutant cell lines through transfection with plasmids into U87 cell lines.3.IDH1-R132 H mutation increased the recruitment of NK cells,contributing to a better prognosis of glioma patients via analyzing TCGA,CGGA databases and clinical tissue samples.4.By analyzing databases,clinical tissue samples and cell lines,we found that IDH1-R132 H mutation up-regulated CX3CL1 expression,thus promoted the recruitment of NK cells.5.The secretion of metabolite 2-HG in IDH1-R132 H mutant cell line promoted the expression of phosphorylated NF kappa B,and thus up-regulated the chemokine CX3CL1 expression.6.We investigated that CX3CL1 was strongly correlated with CD56 expression both in whole glioma patients and IDH1-R132 H mutant glioma patients through analyzing CGGA and TCGA datasets and clinical tissue samples.7.We generated IDH1 knockout U87 cell line by CRISPR-Cas9 tool and contracted IDH1-R132 H homozygous mutant cell line through deleting background expressed IDH1 gene.ConclusionIDH1-R13 H mutation improved prognosis of glioma patients by regulating tumor immune microenvironment.The metabolite 2-HG secreted from IDH1-R132 H mutant glioma promoted p-NF-κB expression and further promoted the expression of chemokine CX3CL1 and thereby recruited more NK cells to the tumor site,leading to a better outcome.In addition,our study provided a new way for NK cell immunotherapy in patients with IDH1-R132 H mutant glioma.